Centre for Tissue Engineering & Regenerative Medicine, Faculty of Medicine, Jalan Yaacob Latif, Cheras, Kuala Lumpur 56000, Malaysia.
Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Cheras, Kuala Lumpur 56000, Malaysia.
Int J Mol Sci. 2023 Feb 13;24(4):3745. doi: 10.3390/ijms24043745.
Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton's Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.
无动物源的三维培养物因其在间充质干细胞（MSCs）扩增中的应用而受到关注。我们研究了无动物源血清替代物，即人血清和人血小板裂解液，在随后的 MSCs 微载体培养中替代当前常规使用的胎牛血清的潜力。在这项研究中，我们将 Wharton's Jelly MSCs 培养在九种不同的培养基组合中，以确定最适合 MSCs 培养的无动物源培养基。我们鉴定了细胞增殖和活力，并按照国际细胞治疗学会（ISCT）定义多能间充质基质细胞的最低标准对培养的 MSCs 进行了特征描述。然后，我们将选定的培养基用于 MSCs 的微载体培养，以确定三维培养系统在未来临床应用中扩增 MSCs 的潜力，并鉴定培养的 MSCs 的免疫调节潜力。低葡萄糖 DMEM（LG）+人血小板（HPL）裂解液培养基似乎是我们单层培养系统中替代传统 MSCs 培养基的良好候选物。在 LG-HPL 培养基中培养的 MSCs 获得了高细胞产量，其特征与 ISCT 描述的一致，尽管细胞的整体线粒体活性低于对照，其后续影响仍不清楚。另一方面，MSC 微载体培养显示出与单层培养相当的细胞特征，但细胞增殖停滞，这可能是由于 FAK 失活所致。尽管如此，MSCs 单层培养和微载体培养均对 TNF-α表现出高抑制活性，只有 MSC 微载体培养对 IL-1 的分泌有更好的抑制作用。总之，我们确定 LG-HPL 是 WJMSCs 培养的良好无动物源培养基，尽管需要进一步的机制研究，但结果表明，无动物源的三维培养保持了 MSC 的特征，并提高了免疫调节活性，这表明将单层培养转化为该培养系统在 MSC 扩增中的潜力，以用于未来的临床应用。
